SCIENCE ADVANCES | RESEARCH ARTICLE

ORGANISMAL BIOLOGY Copyright © 2020 The Authors, some rights reserved; An ancient push-pull pollination mechanism in exclusive licensee Shayla Salzman1,2,3*†, Damon Crook4, James D. Crall1, Robin Hopkins1,2‡, Naomi E. Pierce1,3‡ American Association for the Advancement of Science. No claim to Most cycads engage in brood-site pollination mutualisms, yet the mechanism by which the Cycadales entice original U.S. Government pollination services from diverse insect mutualists remains unknown. Here, we characterize a push-pull pollina- Works. Distributed tion mechanism between a New World and its weevil pollinators that mirrors the mechanism between a under a Creative distantly related Old World cycad and its thrips pollinators. The behavioral convergence between weevils and Commons Attribution thrips, combined with molecular phylogenetic dating and a meta-analysis of thermogenesis and coordinated patterns NonCommercial of volatile attraction and repulsion suggest that a push-pull pollination mutualism strategy is ancestral in this License 4.0 (CC BY-NC). ancient, dioecious group. Hence, it may represent one of the earliest insect/plant pollination mechanisms, arising long before the evolution of visual floral signaling commonly used by flowering .

INTRODUCTION brood-site mutualist is repelled from its host pollen cone and en- Downloaded from Highly specialized coevolved systems are uncommon in nature and ticed into ovulate cones has been described in M. lucida (5), where are sometimes considered evolutionary dead ends (1). However, the Cycadothrips chadwickii thrips (order: Thysanoptera) are attracted plant order Cycadales is one of the oldest living lineages of seed (“pulled”) by lower concentrations of the dominant plant volatile plants (2), and most species form obligate brood-site pollination compound, -myrcene, and repelled (“pushed”) by higher concen- mutualisms with insects (3, 4). Here, we investigate whether unre- trations of the same compound. The daily plant thermogenic and lated pollination mutualists exhibit matching behavioral responses volatile cycle induces pollinator movement between pollen and http://advances.sciencemag.org/ to conserved plant behaviors, representing a shared pollination ovulate cones, enabling pollination and seed set (5). This push-pull strategy. The push-pull pollination mechanism of the Australian pollination mechanism differs from most pollination syndromes cycad Macrozamia lucida is characterized by a daily peak in plant because it has a repulsive component that expels pollinators from thermogenesis and volatile production whereby thrips pollination pollen cones at a certain point in the cycle in addition to the attrac- mutualists are attracted to lower levels of a plant volatile and repelled tive mechanisms commonly seen in the visual and chemical cues of by high levels of the same compound (5). Across the Cycadales, flowers. Furthermore, the same chemical cue is used for attraction pollination-stage cones undergo a daily cycle of thermogenesis (6) and repulsion. and respiratory processes that similarly culminate in a peak of vola- The Mexican cycad furfuracea (Zamiacea), which is more tile production (7). By analyzing a distantly related New World than 150 million years (Ma) diverged from M. lucida, has an obligate cycad species that, like most cycads, is associated with a coleopteran pollination mutualism with Rhopalotria furfuracea (Coleoptera: pollination mutualist, we investigate whether this characteristic Belidae) weevils (formally R. mollis) (17). R. furfuracea complete on September 14, 2020 cycle of thermogenesis and coordinated volatile production is broadly their entire development within the plants’ disposable pollen cone indicative of an ancient and conserved pollination strategy. parenchyma tissue (10), and the plants do not set seed without Within the dioecious Cycadales, specialized insect pollinators, pollinators (17). Specialized coleopteran pollination is widespread mostly beetles and weevils, live and feed almost exclusively within in extant Cycadales (3, 4) and has existed in the lineage since at least the male pollen cone tissue (3, 4, 8–10), although one obligate ovule the mid-Mesozoic where fossilized Boganiidae beetle pollinators parasite may play a minor role in pollination of villosus have been found (18) well before the rise of flowering plants (19) or (9). Successful cycad reproduction requires these brood-site mutu- the earliest known direct evidence of thrips pollination (20). While alists to leave the host pollen cone and transfer pollen to a female push-pull pollination has also been hypothesized for Coleoptera-­ ovulate cone. Over the course of a day, pollination-stage cones pro- pollinated cycads (21, 22), the mechanism by which the plants duce a predictable thermogenic and volatile pattern (7). Both pollen manipulate their behavior to carry out pollination has not yet been and ovulate cones of an individual species produce similar thermo- explored. genic and volatile profiles (6, 11–14), yet across all species, pollen cones produce higher temperature peaks (6) and more concentrated volatile compounds (3, 11, 12, 14). Pollinator exit from pollen cones RESULTS has been observed to coincide with thermogenic and volatile peaks Plant volatile profile and weevil physiological response (5, 12, 15–17). The specific behavioral mechanism by which a We identify the chemical communication mechanism underlying the mutualism between R. furfuracea and Z. furfuracea. Using head- space collection methods, plant volatile compounds were collected 1 Department of Organismic and Evolutionary Biology, Harvard University, 26 Oxford from Z. furfuracea pollen (n = 8) and ovulate cones (n = 7) to deter- Street, Cambridge, MA 02138, USA. 2The Arnold Arboretum, Harvard University, Boston, MA 02131, USA. 3Museum of Comparative Zoology, Harvard University, 26 mine the plant volatile profile and the physiological response of Oxford Street, Cambridge, MA 02138, USA. 4USDA-APHIS-PPQ CPHST, Otis Laboratory, R. furfuracea to host plant volatiles. Z. furfuracea pollen and ovulate Building 1398, Otis ANGB, MA 02542, USA. cones produce two major compounds: the hydrocarbon 1,3-octadiene *Corresponding author. Email: [email protected] and the alcohol linalool (Fig. 1). Electroantenograph detection †Present address: School of Integrative Plant Sciences, Section of Plant Biology, Cornell University, 502 Mann Library, Ithaca, NY 14853, USA. (EAD) demonstrated that R. furfuracea male and female weevils are ‡These authors share senior authorship. physiologically capable of perceiving only 1,3-octadiene (n = 10;

Salzman et al., Sci. Adv. 2020; 6 : eaay6169 12 June 2020 1 of 9 SCIENCE ADVANCES | RESEARCH ARTICLE

A 0.6

0.4 vils in pits

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HO Propor

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5 Fig. 1. Z. furfuracea produce two main volatile components, 1,3-octadiene and linalool, yet R. furfuracea only physiologically respond to 1,3-octadiene. Gas chromatography–flame ionization detector (GC-FID) of Z. furfuracea plant cone volatiles is shown at the top with an example male R. furfuracea weevil electro- 0 antenograph detection (EAD) on the bottom. Scale bar, 1 mV for EAD channel. Red arrow denotes a positive physiological response of weevils to 1,3-octadiene that is http://advances.sciencemag.org/ not seen for linalool (n = 10). Weevil response to 1,3-octadiene and lack of response (mm) sample box ment toward ve –5 to linalool were confirmed with standards (n = 6). Photo credit: Shayla Salzman, Cornell University. evil mo evil We

–10 10,000 650,000 Fig. 1), explaining why previous attempts to elicit behavioral re- ng of 1,3-octadiene sponses in R. furfuracea using linalool were unsuccessful (11). Fig. 2. Weevil behavior follows push-pull pollination where they are attracted Weevil behavioral response to plant volatiles to lower concentrations of 1,3-octadiene and repelled by higher amounts. A Because a physiological response does not equate with attraction ( ) Weevils are preferentially attracted to mid-level amounts of 1,3-octadiene in pitfall tests (shown in fig. S2) and less attracted to more concentrated amounts. on September 14, 2020 and the production of 1,3-octadiene was found to vary over the The proportion of weevils in the bait pit is shown on the y axis. Sequential Bonferonni-­ course of the day (fig. S1), we analyzed the behavioral response of adjusted P values using an ordinary least squares model are found in table S2. R. furfuracea to different amounts of 1,3-octadiene, covering the (B) Weevils are attracted to and move toward 10,000 ng of 1,3-octadiene and are range of intrinsic plant production. Using pitfall tests where groups repelled and move away from that of 650,000 ng in behavioral arenas (shown in fig. S3). of 4 to 10 weevils are introduced for a period of 30 min to a dilution Significance is determined using an ordinary least squares model. Measurements of 1,3-octadiene in dichloromethane or a control (fig. S2), we show above the gray line show a change in average weevil position toward the that R. furfuracea are positively attracted to 1,3-octadiene and that 1,3-­octadiene sample and below the line a shift away from the sample. Individual weevil attraction to increasing amounts of 1,3-octadiene is nonlinear points represent one trial of 4 to 10 weevils in (A) and one trial of ~80 weevils in (B).  (Fig. 2A). Weevils are more attracted to the intermediate amount of Absolute amounts of 1,3-octadiene in 10 l of dichloromethane are shown on the x 10,000 ng of 1,3-octadiene than to all lower quantities (P < 0.012 for axis in both graphs. all lower amounts; table S2) and to the highest quantity (P = 0.0102 for 650,000 ng). The decrease in attraction suggests, but does not Together, these experiments demonstrate that weevil attraction conclusively demonstrate, that weevils are repelled (i.e., pushed) by preference is for an intermediate amount of 1,3-octadiene and that high concentrations. their movement can be induced by varying the quantities of plant We therefore tested whether intermediate (10,000 ng) and high compounds, consistent with the push-pull pollination observed in (650,000 ng) quantities of 1,3-octadiene elicited different movement M. lucida (5). responses in weevils. The push-pull hypothesis predicts that weevils should move toward 10,000 ng of 1,3-octadiene and move away Weevil and plant behavior from 650,000 ng of 1,3-octadiene. We set up enclosed arenas (fig. S3) We assessed the attraction of weevils to pollen microsporophylls that included ~80 weevils, exposing five groups to either 10,000 ng and ovulate megasporophylls (hereafter referred to as pollen and of 1,3-octadiene plus a control or that of 650,000 ng plus a control, ovulate cone scales) in the laboratory over an 8-hour period to look and videotaped their movements over a period of 31 min. We mea- for differences between the pollen cone, which serves as a feeding sured the change in distance toward the 1,3-octadiene sample from site, and the ovulate cone, which is not fed upon. Using small arenas the beginning of the video to the end. As predicted, R. furfuracea containing four weevils with either a pollen cone scale or an ovulate move toward 10,000 ng of 1,3-octadiene but move away from cone scale, we defined “attraction” as a weevil being on or within 650,000 ng of 1,3-octadiene (P = 0.0033; fig. S4 and Fig. 2B). one body length of the cone scale. We find that overall, weevils are

Salzman et al., Sci. Adv. 2020; 6 : eaay6169 12 June 2020 2 of 9 SCIENCE ADVANCES | RESEARCH ARTICLE more attracted to pollen cone scales than ovulate cone scales production of 1,3-octadiene (fig. S1 and Fig. 3C) may act as a “cheating” (P = 0.0177). However, attraction to both pollen and ovulate cone mechanism in that they mimic pollen cones but do not provide food scales changes over the course of the day (P = 0 for pollen cone or a brood site. scales and P = 0.0249 for ovulate cone scales). The decrease in attraction to pollen cone scales in the evening is much greater than the decrease in attraction to ovulate cone scales (test of variance DISCUSSION between pollen and ovulate cone scale attraction, P = 0.0003199) R. furfuracea weevils and C. chadwickii thrips have converged on (Fig. 3A). Because excised cones are known to continue both thermo- the same behavioral response to an ancient plant pollination mecha- genic and volatile patterning (5, 7), we hypothesized that this nism. Both pollinators use the pollen cone as a larval development pattern may be related to plant volatile production and not just the site, as do other known cycad pollinators (3, 4, 11–13). Both insects artificial nature of laboratory experimentation. show differential behavioral responses according to the quantities We therefore investigated the pattern of Z. furfuracea volatile of one host plant volatile compound: attraction to intermediate release and its correlation to R. furfuracea movement in the field. amounts and repulsion to higher amounts. In both cases, an in- We hypothesized that there would be an increase in volatile produc- crease in volatile production correlates with an expulsion of polli- tion in pollen cones and R. furfuracea activity around 19:00 to 20:00 nators from pollen cones, a pattern that has been observed in other after the thermogenic peak at about 18:30 to 19:00 (fig. S4) (6). We cycad genera as well (5, 12, 15–17). In both plants described here, as

collected volatiles from pollination-stage pollen and ovulate cones well as all cycad genera so far tested, the production of key volatile Downloaded from (n = 5; table S1) at hourly intervals and identify a typical diurnal compounds changes throughout the day (Fig. 4). In the Macrozamia/ pattern in volatile production in both sexes of Z. furfuracea, whereby thrips system, this is known to be caused by the increased respira- cones change the ratio of the two major volatile compounds over tion necessary for thermogenesis (7), an ancestral trait among the the course of the day, approaching 100% production of 1,3-octadiene Cycadales (6), that by itself (22) or together with associated changes in the evening (Fig. 3B). Pollen cones consistently produce a greater in humidity likely acts as an additional cue. quantity of volatile compounds (P = 0.0007) than ovulate cones and The Cycadales are the basal gymnosperm lineage (23) and have http://advances.sciencemag.org/ show a clear pattern of increase in production of 1,3-octadiene in an extensive fossil record stretching back 265 Ma (24), placing them the evening (P = 0.0184; Fig. 3C) that coincides with R. furfuracea among the most ancient extant seed plants. Specialized pollination expulsion from pollen cones around 20:00 (Fig. 3D). Ovulate cone has existed in this group since well before the rise of flowering

A Ovulate cone scale B Ovulate cone 1,3-Octadiene Pollen cone scale Pollen cone Linalool

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60 75 on September 14, 2020

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–250 0 Number of we 1,3-Octadiene percent increase 0 15:00 17:50 20:00 12:50 15:00 17:50 20:00 12:50 15:00 17:50 20:00 Time of day Time of day

Fig. 3. An increase in production of 1,3-octadiene by Z. furfuracea is positively correlated with an exodus of R. furfuracea weevils from the pollen cone. (A) Weevils are more attracted to pollen cone scales, but the percent of weevils on pollen cone scales drops sharply over time in laboratory behavioral arenas. The smoothed condi- tional mean is shown (method = loess) with one SE at 0.95 confidence in (A) to (C), and significance is determined using a weighted least squares model. Mean values with one SE at 0.95 confidence are overlaid in (A). Raw values are overlaid in (B) and (C). (B) The percent composition of the daily volatile profile shifts toward 100% 1,3-­octadiene after 20:00 hours in both pollen and ovulate cones. (C) The percent increase in 1,3-octadiene production in pollen cones rapidly increases after 20:00 hours. (D) Weevils housed in pollen cones are repelled from the cones at 20:00 hours. Each colored line represents an individual pollen cone, and numbers are raw counts of weevils seen in video recordings.

Salzman et al., Sci. Adv. 2020; 6 : eaay6169 12 June 2020 3 of 9 SCIENCE ADVANCES | RESEARCH ARTICLE

? ? Cycas (117) ring plants ? ? we (16)

? ? Rise of flo (2)

Macrozamia (41)

? ? Lepidozamia (2) Coleoptera pollinator

Lepidoptera pollinator ? Encephalartos (65) Thysanoptera pollinator Downloaded from Daily thermogenic pattern Daily volatile pattern ? ? Push-pull pollination described (31)

Stangeria ? ? http://advances.sciencemag.org/ (1) Specialized beetle pollinator fossil (167 Ma) ? ? (1) Fossil evidence of potential thrips pollination (110 Ma) Zamia Pollinivory fossil (245 Ma) Molecular dating of (79) weevil pollinator evolutions This study 350.0 300.0 250.0 200.0 150.0 100.0 50.0 0.0 Carboniferous Permian Triassic Jurassic Cretaceous Tertiary Paleozoic Mesozoic Cenozoic

Fig. 4. Plant behaviors responsible for push-pull pollination are widespread in Cycadales. Bayesian molecular-dated phylogeny of Cycadales shows all known infor- mation on pollinators and thermogenic and volatile patterns. The number of species in each genus is noted in parentheses. Gray bars show the 95% confidence interval on September 14, 2020 for dating at the nodes. Fossil evidence of cycad pollination, molecular dating of weevils pollinator lineages (41), and the estimated timing of the rise of flowering plants are noted. Fossil evidence is as follows: Recently reported fossil of a specialized Boganiidae beetle pollinator (18), the earliest evidence of thrips associated with cycad pollen (20) and the earliest evidence of cycad pollenivory in the form of pollen filled coprolites, likely of beetle origin (25). Citations for metadata are provided in table S3. plants (19) from at least the mid-Mesozoic (18) and perhaps as early tive volatile compounds in cycad push-pull pollination maintain a as 245 Ma (Fig. 4) (25). Stereotypical sequential timing of thermo- repellent component for their specialized mutualists. Early insects genesis in pollen and ovulate cones and specialized brood-site polli- are thought to have found rewards such as high concentrations of nation have been documented to occur across the Cycadales (table S3). starch in cone tissues (4) and pollination drops (4, 31), ovular These observations, in combination with our discovery here of con- exudates found in cycads and other gymnosperms that predate vergence in the behavioral responses of both thrips and weevils to compositionally similar angiosperm nectar. If early cycad herbivores volatiles released by the cones of Old World (Macrozamia) and overcame any deterrent quality of host plant compounds and found New World (Zamia) lineages, support the hypothesis that the push- brood sites in plant reproductive tissues, then plant volatiles may pull system found in cycads represents the most ancient insect/ have become the basis of chemical plant-insect communication, plant pollination mechanism yet documented. Plant thermogenic with signals for attraction and repulsion mediated, in part, by the behaviors appear to have remained consistent over evolutionary daily cycle of thermogenesis. time although extant insect pollinators may represent more recent shifts (26, 27). These dioecious plants give us insight into the earliest forms of insect pollination before the prolific diversification of visual MATERIALS AND METHODS floral signaling and reward systems used by angiosperms. Odor Experimental design attraction has been thought to precede color attraction in angio- The purpose of this study was to determine the insect and plant sperms (28), and the coupling of thermogenesis with strong odors behaviors responsible for the specific brood-site pollination mutualism as seen in cycads has been suggested as an exaptation, leading to seen in Z. furfuracea cycads and their weevil pollinators, R. furfuracea, early angiosperm pollination syndromes (29). Plant volatiles attrac- and to contrast that with the behaviors seen in other cycad pollina- tive to pollinators have been hypothesized to be derived from herbivore tion systems, including M. lucida cycads and their thrips pollina- deterrent secondary compounds (30), and here, we find that attrac- tors, Cycadothrips chadwicki (5). Plant volatile analysis was used to

Salzman et al., Sci. Adv. 2020; 6 : eaay6169 12 June 2020 4 of 9 SCIENCE ADVANCES | RESEARCH ARTICLE identify the volatile components produced by pollen and ovulate mass-selective detector. The GC was equipped with a DB-5 column cones. Physiological analysis of R. furfuracea weevils was used to [J&W Scientific Inc., Folsom CA; 30 m by 0.25 mm inside diameter identify the electro-antennally active plant compounds and behavioral (ID); film thickness, 0.25 m; splitless mode]. Helium was the carrier responses of R. furfuracea to plant compounds. Field experiments gas at a constant flow rate of 0.7 ml/min. The injector temperature matched plant volatiles corresponding to weevil behavior to confirm was 250°C. Oven temperature was held at 40°C for 1 min, pro- laboratory experiments. Last, thermogenic and volatile patterns grammed to 170°C at 10°C/min, and held for 5 min. Volatile peaks across cycad genera were mapped onto a dated phylogeny to pro- were manually integrated. Volatiles were identified on the basis of vide an evolutionary context. their mass spectra (NIST version 2.0, 2002), Kovats indices (34, 35), and by comparison of the retention indices and mass spectra with Study species those of available authentic synthetic compounds and a computer- Z. furfuracea is an endangered dioecious gymnosperm native to ized data library (NIST version 2.0,2002). An authentic standard of Veracruz, Mexico (32). Both pollen and ovulate cones are known to 1,3-octadiene was obtained from ChemSampco Inc. (Dallas, TX, undergo a daily synchronous thermogenic peak at the time that catalog no. 7015.90). Authentic standards of (±) linalool were obtained they are reproductive, with a higher temperature spike in pollen from Sigma-Aldrich Co. (Saint Louis, MO, catalog no. L2602-5G). cones (2.5°C above ambient; ovulate cones, 0.8°C above ambient) (6). Pollen cones produce two main volatile components, the Electrophysiological analysis: GC-EAD

hydrocarbon 1,3-octadiene and the alcohol linalool (11). The physiological response of R. furfuracea weevils to host plant Downloaded from R. furfuracea is an obligate pollination mutualist with Z. furfuracea Z. furfuracea volatile compounds was determined using GC-EAD. (10, 17). It is required for Z. furfuracea reproduction (17) and its The coupled GC-EAD system used was as previously described by entire life cycle is tied to that of its host plant (10). R. furfuracea Crook et al. (36), with a few modifications. Samples of aerations or adults feed, breed, and lay eggs in Z. furfuracea pollen cones and standards were injected (2 l), splitless, onto a Hewlett Packard larvae feed and develop inside of microsporphylls, pupating inside (Agilent Technologies, Santa Clara, CA) 6890 GC with a DB-5MS- of the microsporophyll stalk (10). R. furfuracea visit ovulate cones DG column (J&W Scientific Inc., Folsom, CA; 0 m by 0.25 mm ID, http://advances.sciencemag.org/ of Z. furfuracea but do not feed or lay eggs on them. The life cycle is 0.25-m film thickness) and a 1:1 effluent splitter that allowed si- 7 to 8 days, with many generations occurring during the plant multaneous flame ionization detector (FID) and EAD detection of reproductive season (10). At the end of the plant reproductive the separated volatile compounds. Helium was the carrier gas season, late instar larvae go into diapause for the 10 months of the (2.5 ml/min). Oven temperature was held at 50°C for 1 min, pro- year when plants are not reproductive, remaining inside of the grammed to 280°C at 10°C/min, and held for 15 min. Injector tem- stalks of the dried, dead microsporphylls (10). perature was 280°C. The GC outlets for the EAD and FID were 300°C. The column outlet for the EAD was held in a water-cooled Volatile collection humidified air stream (20°C) flowing at 20 ml/min over the antennal Z. furfuracea plant volatile collections were performed using head- preparation of adult R. furfuracea attached to an EAG probe space collection methods (33) on eight pollen dehiscing male cones (Syntech, Hilversum, the Netherlands). Whole heads of an adult and seven receptive female cones to determine the volatile profile. beetle were removed so that both antennae could be used for re- on September 14, 2020 Three pollen cones and two ovulate cones were further sampled at cording. Tips of both antennae were cut off to make a clean opening hourly time points throughout the day to assess daily variation. All for conducting gel (Spectra 360, Parker Laboratories, Fairfield, NJ) volatile collections were done in situ at Montgomery Botanical to form an uninterrupted connection to the EAG probe. The EAG Center in Miami, Florida. Accession information for these plants is probe was connected to an IDAC-232 serial data acquisition con- found in table S1. Cones remained attached to the plant and cov- troller (Syntech). Signals were stored and analyzed on a PC equipped ered in oven bags (Reynolds Consumer Products, Lake Forest, IL) with the program EAD (version 2.6, Syntech). GC-EADs using whole that were tied shut at the bottom of the cone. Low-flow vacuum air Z. furfuracea plant volatile collections were performed on six males samplers (Gilian model LFS-113DC) calibrated to 0.1-liter/min and four females, and GC-EADs of the 1,3-octadiene standard were pulled headspace air and volatiles for 45 min through filters made performed on three males and three females. with 300-mg Porapak Q adsorbent mesh 80-100 75 cm3 (Sigma-Aldrich, Saint Louis, MO, part no. 20331) held into glass Pasteur pipettes Behavioral analysis: Pitfall tests of 1,3-octadiene size 14.6 cm (VWR International, Radnor, PA, part no. 14672-200) with concentrations plugs of glass wool (Sigma-Aldrich, Saint Louis, MO, part no. 20411). Because the GC-EAD is only able to determine a physiological Before volatile collection, filters were soaked in high-performance response, the behavioral response (attraction or repulsion) of liquid chromatography (HPLC)–grade dichlormethane (Sigma-­ R. furfuracea weevils to the Z. furfuracea plant volatile compound Aldrich, Saint Louis, MO, part no. 650463) for 48 hours and then 1,3-octadiene was determined in pitfall tests (fig. S2) using differing prewashed 10 times with full flow-throughs of dichloromethane amounts of the compound, along with a control of dichloromethane and dried with charcoal-purified air pushed through at 0.4 liter/min. carrier solvent. Pits were constructed of 100 mm by 15 mm petri After volatile collection, samples were eluted with 500 l of HPLC- dishes with 1.5-cm circular holes cut into them and hot glued onto grade dichloromethane (Sigma-Aldrich, Saint Louis, MO, part no. 5-cm-deep plastic cups. These were set into larger glass cups so that 650463) pushed through with charcoal-purified air. all dishes were completely level. Ten microliters of 1,3-octadiene standards diluted in dichloromethane (1, 10, 100, 1, 10, and Gas chromatography–mass spectrometry 65.5 g/l) was applied to 1 cm by 4 cm pieces of filter paper (Whatman Initial chemical analyses were conducted using a combined Agilent plc, no. 4) (GE Healthcare Life Sciences, Chicago, IL) handled with Technologies 6890 network gas chromatograph (GC) and 5973 forceps and dropped into the pits. For each trial, 1,3-octadiene

Salzman et al., Sci. Adv. 2020; 6 : eaay6169 12 June 2020 5 of 9 SCIENCE ADVANCES | RESEARCH ARTICLE concentrations and controls of 10 l of dichloromethane were run each identified weevil, distance was calculated to the nearest point simultaneously. The number of trials for each treatment is as follows: on each bait. If weevils were located within the edges of the baits, dichloromethane control, 28 trials; 10 ng, 26 trials; 100 ng, 26 trials; then the distance to that bait was set to zero. For each sampled 1000 ng, 27 trials; 10,000 ng, 27 trials; 100,000 ng, 26 trials; and frame, a mean distance to sample and to control was calculated 650,000 ng, 22 trials. R. furfuracea weevils were taken directly from across all the weevils tracked for that frame, giving one mean value Z. furfuracea pollen cones so as to be well fed. Five to 10 weevils for the group. were placed inside the edge of the petri dish, which was then closed Weevil movement in relation to 1,3-octadiene amount was and placed in the dark at room temperature (22°C) for 30 min, at determined by the change in distance to the sample from the start which time the number of weevils inside of the pits was counted and of the trial to the end. The “starting” distance to the sample box was transformed into proportions (weevils in pits/total weevils). Dead determined for each trial by creating one mean value of weevil loca- weevils or copulating weevils were not counted. tion over the first 5 min of video. Weevils were then given ~18 min to make a choice, and before the “ending,” asymmetry toward Behavioral analysis: Weevil movement in relation sample was determined, beginning at ~23 min (sampled frame, no. 700). to 1,3-octadiene concentrations On the basis of personal observations, 18 min provides sufficient To determine whether the high concentration of 1,3-octadiene acts time for weevil activity to begin, and the results were not sensi- as a repellant, arenas were constructed in which 80 to 100 R. furfuracea tive to this specific cutoff. Autocorrelation of the change in distance

weevils were introduced, and their behavior was recorded for to the sample was analyzed using the acf function in R separately for Downloaded from 31 min in a series of five trials to either 10,000 ng (n = 2) or each trial. We then computed the average autocorrelation function, 650,000 ng of 1,3-octadiene (n = 3) suspended in dichloromethane. which showed a substantial diminishment in autocorrelation Arenas were constructed of white acrylic sides and bottom with a (correlation, <0.1) at a lag of 2 min (60 sampled frames) across all clear acrylic top (fig. S3). Dimensions were 14.5 cm by 9.5 cm and videos. We therefore subsampled our raw tracking data by taking a 3.5 cm tall. To allow airflow and the dissipation of volatile com- 1-min (30 sampled frames) mean value for weevil location every pounds, windows were cut into the sides and covered with white 3.33 min (100 sampled frames) to minimize the impacts of autocor- http://advances.sciencemag.org/ mesh fabric (13.5 cm by 1 cm holes in the long sides, and 8.2 cm by relation. The ending distance to sample was then subtracted from 1 cm holes in the short sides). Openings (2 cm by 5 cm) were cut in the starting distance to sample (n = 6 for 10,000 ng and n = 9 for the bottom of the arena for sample and control. Weevils for each 650,000 ng of 1,3-octadiene). trial were inserted through these openings, which were then covered with fine white mesh fabric from the bottom. Ten microliters of Behavioral analysis: Weevil attraction to micro- 1,3-octadiene dilution (1 or 65.5 g/l) in dichloromethane was and megasprophylls placed onto 1 cm by 4 cm filter paper (Whatman plc, no. 4) (GE Weevil attraction to pollen and ovulate cones over the course of a Healthcare Life Sciences, Chicago, IL) and set under the opening in day was determined through video analysis of small arenas contain- the bottom of the arena. To account for any visual attraction to the ing four weevils and either a pollen cone microsporophyll or an openings in the arena floor, a blank filter paper piece was placed ovulate cone megasporophyll (pollen and ovulate cone scales). Pollen on September 14, 2020 under the other opening as a control. This entire setup was placed and ovulate cone scales were collected from pollination-stage cones on a clear acrylic panel suspended over a 29.6 cm by 29.6 cm at Montgomery Botanical Center in Miami, Florida. Four ovulate light-emitting diode (LED) light panel (no. BK3301, U.S. Solid State and two pollen cones were stored at 16°C overnight beginning at LLC, Shreveport, LA) and encased in a black box covered with black 21:30 hours. They were removed at 8:00 hours and slowly brought fabric so as to be backlit. Weevils were allowed to feed freely on Z. up to 82°C over the course of 1 hour. Video analysis began 1 hour furfuracea pollen cones before trials so as to be well fed. later at 10:00 hours. Thirty-two trials were conducted simultaneously Raw videos were recorded using a Sony Handycam HDR-CX260V with 16 replications of pollen cone scales and 16 replications of (Sony, Tokyo, Japan) and subsequently processed in MATLAB ovulate cone scales arranged so as to have as much separation be- using custom scripts. The locations of the outer arena edges and the tween individual cones and sexes as possible. Sample map is shown in left and right bait locations were manually located for each trial. fig. S5. Arenas were set up as follows: 60 mm by 15 mm petri dishes Next, a background image was computed separately for each trial by were place upside down on top of filter paper (Whatman plc, no. 4) calculating the median intensity at each pixel. This median-averaging (GE Healthcare Life Sciences, Chicago, IL), creating an enclosed method meant that weevils that were mostly immobile are considered environment. This setup rested on top of wide plastic mesh to allow part of the background and excluded from further analysis. for airflow and dissipation of any plant volatile compounds from For each 31-min trial, where 80 to 100 weevils were introduced the arena. Cone scales were broken off of the cone at 9:30 hours and to either amount of 1,3-octadiene, we analyzed a subset of 930 frames placed in the middle of the designated arena. Four R. furfuracea (video frame rate, 29.97 frames/s; subsampling, 1 frame every 2 s). weevils were taken directly from pollen cones so as to be well For each analyzed frame, weevils were separated from the back- fed and placed into each arena. The entire setup was placed into ground by subtracting the frame’s pixel intensity values from the a white plastic box that was illuminated from all four sides with computed background image. This intensity-differential image was fluorescent lighting and covered with black cloth so as to be evenly then turned into a binary map of weevils using a manually calibrated lit. Videos were recorded using a Sony HandyCam HDR-CX260V intensity threshold. After this segmentation step (and digital ero- (Sony, Tokyo, Japan) and began at 10:01 hours and ended at sion and dilation to reduce noise), image regions that were within a 21:30 hours. Weevils’ attraction to cone scales was manually size threshold (i.e., between 150 and 800 pixels, manually calibrated counted from the resulting video at 30-min intervals. Weevils to exclude digital noise) and located within the arena were consid- were counted as attracted to the cone if they were within 1 mm of ered separate weevils (average weevil pixel area = 300 pixels). For the cone.

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Cone thermogenesis between groups and an ordinary least squares model (P = 0.1044). Z. furfuracea thermogenesis patterns were tested on in situ cones at Therefore, the ordinary least squares model with fewer parame- Montgomery Botanical Center in Miami, Florida. One pollination-­ ters was used to determine significant differences in proportions of stage ovulate cone, one pollination-stage pollen cone, and one weevils attracted to baits between the different amounts of prepollination-stage pollen cone were tested and compared with 1,3-octadiene [182 degrees of freedom, t values = 0.148 (10 ng/l), published data (6). Temperature probes from Onset UX120-014M −0.0157 (100 ng/l), 0.283 (1000 ng/l), 3.789 (10,000 ng/l), 2.172 (Onset, Bourne, MA) data loggers were inserted into the cone (100,000 ng/l), and 0.0458 (650,000 ng/l)]. For behavioral arenas, peduncles between mega- or microsporophylls and recorded from a likelihood ratio test was used to determine whether variances 8:00 until 21:00. Ambient temperature was collected adjacent to the between the groups should be accounted for, and no significant plants. The plants, including the ambient temperature probe, were difference was found between a weighted least squares model that covered with shade cloth to prevent direct heating from the sun. allowed for different variances between groups and an ordinary least squares model (P = 0.5869). Therefore, the ordinary least Weevil expulsion from male cones squares model with fewer parameters was used to determine the Ten pollen dehiscing male cones were collected and videotaped significance of change in distance to sample for the two amounts of throughout the course of the day in conjunction with hourly plant 1,3-octadiene (t value = −3.596, 15 degrees of freedom). For weevil volatile collections to determine the correlation between plant attractiveness to cone scales, weighted least squares models were

volatile emission and weevil expulsion from male cones. Cones used to account for variation between groups in determining the Downloaded from were placed into 20.5 cm by 17 cm by 17 cm clear plastic containers. significance between pollen and ovulate cone attractiveness (t value = One square side of the container was mesh to allow airflow and 2.379, 512 degrees of freedom). For attractiveness over time, no sig- volatile dissipation, and the cone was placed closest to this side. nificant difference was found between a weighted least squares Cones used in video analysis were the same stage as those used for model and an ordinary least squares model (P = 0.9853 for pollen volatile collection, open and releasing pollen. These boxes were cone scales and P = 0.9558 for ovulate cone scales) so the ordinary placed on top of a white sheet of paper set on a clear plastic sheet least squares with fewer parameters was used to determine signifi- http://advances.sciencemag.org/ that was suspended over a 29.6 cm by 29.6 cm LED light panel cance (t value = −5.0315, 256 degrees of freedom for pollen cone (no. BK3301, U.S. Solid State LLC, Shreveport, LA). This entire setup scales; t value = −2.256, 256 degrees of freedom for ovulate cone was covered with black cloth so that the only source of light was the scales). Differences in the variances in attractiveness between pollen even backlit LED panel. Cones were videotaped for 11 hours using and ovulate cone scales were determined using a variance test. Last, a Sony Handycam HDR-CX260V (Sony, Tokyo, Japan). Weevil differences in volatile production between pollen and ovulate cones expulsion from the cone was manually counted at 30-min intervals were determined using a weighted least squares model to account from the resulting videos. for the variation between cone sexes (t value = 3.595, 56 degrees of freedom), and differences in 1,3-octadiene increases over time in Phylogeny pollen cones were determined using an ordinary least squares model Dated molecular phylogenetic analyses have been performed in the (t value = 2.566, 22 degrees of freedom) when no significant differ- Cycadales with similar dates but conflicting generic relationships ence was found between a weighted least squares model and an on September 14, 2020 (37–39). The dataset from Salas-Leiva et al. (38) was used to con- ordinary least squares model (P = .6334). struct a dated phylogeny using the methods and “traditional” fossil calibrations suggested in Condamine et al. (37). The Salas-Levia et al. SUPPLEMENTARY MATERIALS dataset (38) was chosen over Condamine et al. (37) because of its Supplementary material for this article is available at http://advances.sciencemag.org/cgi/ use of markers from more gene regions (5 versus 3) and the com- content/full/6/24/eaay6169/DC1 pleteness of its matrix (100% gene coverage for all taxa versus 51% View/request a protocol for this paper from Bio-protocol. coverage). The two datasets were not combined because the repre- sented taxa did not overlap completely, and preference was given to REFERENCES AND NOTES a complete data matrix. Molecular dating was performed using 1. J. C. Vamosi, W. S. Armbruster, S. S. Renner, Evolutionary ecology of specialization: Insights from phylogenetic analysis. Proc. R. Soc. Lond. Biol. 281, 20142004 (2014). BEAST v.1.10.0 (40). Fossil calibrations are based on Hermsen et al. 2. S.-M. Chaw, C. L. Parkinson, Y. Cheng, T. M. Vincent, J. D. 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61. T. E. Marler, K. J. Niklas, Reproductive effort and success of Cycas micronesica K.D. Hill are by the National Science Foundation Physics of Living Systems program (1411123). Published affected by habitat. Int. J. Plant Sci. 172, 700–706 (2011). by a grant from the Wetmore Colles Fund. Author contributions: S.S., N.E.P., and R.H. 62. W. Tang, Heat production in male cones of seven Asian Cycas species (Cycadales), in conceived of the project; S.S. performed all field and behavioral experiments, GCMS, and Cycad biology and conservations: The 9th international conference on cycad biology (New statistical analysis; D.C. performed EAD analysis, S.S. and J.D.C. designed the behavioral arena York Botanical Garden, Bronx, NY, 2019), pp. 486–496. experiment; and J.D.C. performed the video analysis. Competing interests: The authors 63. I. Terry, C. Calonje, M. A. Calonje, T. E. Marler, Thermogenesis patterns in selected Cycas declare that they have no competing interests. Data and materials availability: All data species, in Cycad biology and conservations: The 9th international conference on cycad needed to evaluate the conclusions in the paper are present in the paper and/or the biology (New York Botanical Garden, Bronx, NY, 2019), pp. 410–432. Supplementary Materials. All data are freely available on Harvard Dataverse: https://dataverse. harvard.edu/dataverse/ZP. Additional data related to this paper may be requested from the Acknowledgments: We thank I. Terry for the use of air sampling pumps and guidance in authors. volatile collection and Montgomery Botanical Center for access to collections and the help of knowledgeable staff, especially M. Calonje and P. Griffith. S.S. thanks the thesis committee, Submitted 3 July 2019 including B. Farrell, D. Haig, and D. Stevenson as well as M. Whitaker, S. Worthington, Accepted 17 April 2020 A. Cossée, and two anonymous reviewers for advice on methods and/or comments on the Published 12 June 2020 manuscript and R. Maher for making data collection possible. Funding: S.S. was supported by 10.1126/sciadv.aay6169 a National Science Foundation Graduate Research Fellowship (NSF GRFP) and is now supported by an NSF Postdoctoral Research Fellowship in Biology (1906333), J.D.C. is Citation: S. Salzman, D. Crook, J. D. Crall, R. Hopkins, N. E. Pierce, An ancient push-pull supported by the Winslow Foundation and the Rockefeller Foundation, N.E.P. was supported pollination mechanism in cycads. Sci. Adv. 6, eaay6169 (2020). Downloaded from http://advances.sciencemag.org/ on September 14, 2020

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